Controlling reactogenicity while preserving immunogenicity from a self-amplifying RNA vaccine by modulating nucleocytoplasmic transport

Figures

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  Fig. 1. RNAx enhances GOI expression from saRNA in vitro. A The Cardiovirus leader protein suppresses innate signaling by dampening nucleocytoplasmic transport (NCT) in infected cells. RNAx refers to an mRNA that encodes for the leader protein. B RNAx was expressed from VEEV saRNA in cis from an IRES downstream of GOI or added in trans from a modified nucleoside mRNA. C RNAx enhances GOI expression in cells with intact innate signaling. BJ or 293 T cells were transfected with reporter constructs and Luc signal measured 48 h later with or without RNAx in cis. D RNAx enhances GOI expression in a dose-responsive fashion in trans. BJ cells were transfected with secreting nLuc-expressing saRNA with increasing amounts of RNAx in trans and 6 days post transfection supernatants were harvested for nLuc activity. E RNAx reverses suppression of mRNA reporter activity in the presence of IFN-β or dsRNA (Poly I:C). BJ cells were transfected with a modified nucleoside fLuc-encoding mRNA and co-treated with either 1 ng/mL recombinant IFN-β or 100 ng/mL Poly(I:C). Luc activity was measured 48 h post transfection from cell lysates. Mean ± SEM, n = 3–4. *p < 0.05, **p < 0.01, ****p < 0.0001 Ratio paired…

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  Fig. 2. RNAx enhances GOI expression from saRNA in vivo. A Unmodified Influenza HA-nLuc expressing saRNA was co-formulated with modified nucleoside RNAx mRNA (in trans) or co-expressed downstream of an IRES (in cis) and packaged into LNPs. ‘In trans’ co-formulations consisted of at 36% (weight/weight) RNAx mRNA and 64% saRNA i.e., 0.72 µg of RNAx and 1.28 µg saRNA. B Flow cytometry of Influenza HA surface expression in 293 T cells treated for 24 h with HA-nLuc saRNA-LNPs (no RNAx) at the indicated concentrations. C C57BL/6 mice (n = 5 per group) were intramuscularly injected with 2 µg Influenza HA-nLuc expressing saRNA-LNPs and nLuc activity at the injection site quantified with IVIS on the indicated days post injection. D Total flux of Luc signal is represented by photons per sec (p/s). Quantification of nLuc activity at the injection site quantified with IVIS on the indicated days post injection or the area under the curve (AUC). Geometric mean ± Geometric SD, **p < 0.01, ***p < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons. Numbers above bars indicate average fold change enhancement of RNAx containing groups relative to saRNA only. Created with BioRender.com.

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  Fig. 3. RNAx suppresses the expression of proinflammatory cytokines and enhances GOI expression in human PBMC. A Unmodified Influenza HA-nLuc expressing saRNA was co-formulated with modified nucleoside RNAx mRNA (in trans) or co-expressed downstream of an IRES (in cis) and packaged into LNPs. ‘In trans’ co-formulations consisted of at 36% (weight/weight) RNAx mRNA and 64% saRNA i.e., 0.72 µg of RNAx and 1.28 µg saRNA. B Human PBMC (n = 4 donors) were treated with HA-nLuc saRNA-LNPs for 24 h and supernatants harvested for cytokines and cell lysates for nLuc activity. C The induction of 48 cytokines was measured from supernatants of PBMC treated with 5 µg/mL saRNA-LNPs. Cytokines induced by saRNA alone were determined by comparing the relative levels (ratio of geometric means) between saRNA without RNAx and untreated PBMC. Dotted lines = p < 0.05 (ratio paired t-test) and fold change = 2. D Among saRNA-LNP-responsive cytokines, the relative impact (ratio of geometric means) of RNAx in cis or trans with saRNA LNPs was determined with the ratio paired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). E Supernatants from the same experiment were independently analyzed for IFN-α by ELISA and F…

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  Fig. 4. RNAx suppresses proinflammatory cytokines in mice following vaccination. A 5mC-modified Influenza HA expressing saRNA was co-formulated with modified nucleoside RNAx mRNA in trans at 1% and 10% (weight/weight) into LNPs. B Flow cytometry of Influenza HA surface expression in 293 T cells treated for 24 h with HA saRNA-LNPs (no RNAx) at the indicated concentrations. C C57BL/6 mice (n = 5 per group) were intramuscularly injected with 0.5 µg of Influenza HA saRNA-LNPs and were analyzed for serum cytokines 6 h post injection following (D–F) the prime on day 1 or (G–I) the boost on day 28. D, G Cytokines induced by saRNA only containing LNPs were determined by comparing the relative levels (ratio of geometric means) between saRNA alone and vehicle treated animals. Dotted line = p < 0.05 (Mann–Whitney t-test). E, H Among saRNA-LNP-responsive cytokines, the relative impact (ratio of geometric means) of co-formulating 1% or 10% (w/w) RNAx with saRNA LNPs was determined with Kruskal-Wallis test with Dunn’s multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001) following the prime and boost, respectively. F, I Absolute IFN-α abundance levels for all treatment groups following the prime…

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  Fig. 5. Impact of RNAx on immunogenicity from an Influenza HA saRNA-LNP vaccine. A C57BL/6 mice (n = 5 per group) were intramuscularly injected (prime and boost) with 0.05 µg or 0.5 µg of HA saRNA-LNPs (see Fig. 4A for details on RNA co-formulations). Serum was collected 6 h after the boost on day 28 (post prime measurement) and serum and splenocytes collected on day 42 (post boost measurement). Splenocytes were treated with 1 µg/mL Influenza HA peptide pools and analyzed for B IFN-γ and C IL-4 secreting cells by ELISPOT. Spot forming cells (SFC) were subtracted by untreated samples and normalized by 106 splenocytes. Mean ± SEM. Total anti-HA IgG in the serum (EC50) was quantified by ELISA at D post prime and E post boost. F Neutralization titers were quantified by HAI assay post boost. Dotted line = lower limit of quantification, Geometric mean ± Geometric SD, *p < 0.05, Kruskal-Wallis test with Dunn’s multiple comparisons. Numbers above bars indicate fold enhancement of RNAx containing groups relative to saRNA only. Created with BioRender.com.